Field of the Invention
This invention relates to a process for producing thymosin .alpha..sub.1. More particularly, this invention is directed to a solid-phase synthesis for thymosin .alpha..sub.1 wherein the alpha amino acid of each successive amino acid is protected by 4-methoxybenzyloxycarbonyl (Moz) protective group followed by acidolytic catalyzed cleavage.
Biologically active peptides generally exhibit their biological activity at very minute dosages, for example, at nanogram levels or lower. Therefore, during the initial screening and testing phases it is generally sufficient to produce the peptide of interest in correspondingly small amounts, such as from a few milligrams to a few grams. In such cases, the volume of liquid reagents required during the synthesis and purification steps as solvents, reactants, diluents, washing agents and the like, while often considerable, for example, from several to tens of gallons, nevertheless, may still be manageable in terms of cost, storage space, disposal and the like. However, for large scale production, for example, in lot sizes of from tens of grams or more, for example, from about 100 grams to 2 kilograms, especially 250 grams to 1 kilogram (on dry basis) the problems of product recovery, product purity, and reagent handling, storage and disposal have presented such difficulties that there are virtually no satisfactory large scale processes available. As a result there is a great need in the art for a solid-phase peptide synthesis process capable of producing peptide materials of commercial interest, whether for large scale clinical trials or for other commercial applications, in large batch quantities.
This need is even more urgent for relatively long peptides, i.e., of from about 10 or more amino acids in the peptide, especially from about 15 to 50 amino acid sequences. For example, even using an automated solid-phase peptide synthesizer machine, each full cycle of protection of functional groups, coupling, cleavage of peptide from resin, purification and so on will take from one day to several days or weeks, with the total production process requiring as long as up to 6 to 8 months in some cases. Since this time period is largely independent of batch size it can be readily appreciated that, on a unit basis, production costs are extremely high, when only small gram quantities are produced.
In U.S. Pat. No. 4,079,127, thymosin .alpha..sub.1 is shown to have a molecular weight of 3,108 and a pI' in the range of 4.0-4.3 as determined by slab gel isoelectric focusing at a pH range of 3-5. The compound has the following amino acid sequence:
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu- Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH. PA1 "[g]iven the overall status of current methodology, N.sup..alpha. -protection with the benzyloxy group will probably be restricted to the NH.sub.2 -terminal residue of a peptide. The widely applied protection scheme for solution synthesis, relying on the hydrogenolyzable N.sup..alpha. -benzyloxycarbonyl group and acidolyzable side-chain tert-butyl derivatives . . . has not been applicable for solid-phase synthesis." PA1 "groups are primary urethanes that are deprotected under the same acidolytic conditions and at essentially the same rates as the tert-butyloxycarbonyl groups . . . Hence, they have occasionally been used in stepwise solid-phase synthesis. The ultraviolet absorbing chromophore of [Moz] facilitates spectrophotometric monitoring of deprotection and coupling steps." PA1 (a) temporarily chemically protecting the reactive amino group at the alpha-position and any other reactive groups, other than the carboxylic acid group at the beta-position, on the C-terminal amino acid of the thymosin .alpha..sub.1 peptide; PA1 (b) chemically bonding the protected C-terminal amino acid via the carboxylic acid (--COOH) group thereof to a resin support; PA1 (c) chemically deprotecting the reactive amino group of the resin-bound protected amino acid by acidolytic cleavage using dilute trifluoroacetic acid solution; PA1 (d) chemically coupling via a peptide bond the next amino acid in the desired sequence by contacting the resin-bound amino acid from step (c) with said next amino acid having all of the reactive groups thereof, other than the carboxylic acid group at the alpha-position, chemically protected, in the presence of a coupling agent; PA1 (e) chemically deprotecting the reactive amino group of the coupled amino acid from step (d) by acidolytic cleavage using dilute trifluoroacetic acid solution; PA1 (f) continuing the synthesis by repeating steps (d) and (e) with successive amino acids in the desired sequence being added one at a time until the total desired sequence of the protected peptide is built up on the resin, and PA1 (g) cleaving the protected peptide from the resin support and deprotecting the protected side-chain reactive groups; PA1 wherein the volume of trifluoroacetic acid solvent is substantially reduced by temporarily chemically protecting the alpha-amino acid in step (a) and in step (D) with 4-methoxybenzyloxycarbonyl.
Thymosin .alpha..sub.1 has been found to be 10 to 1,000 times more active than thymosin fraction 5 in several in vitro and in vivo assay systems designed to measure T-cell differentiation and function. Thymosin .alpha..sub.1 may be administered to warm blooded mammals by parenteral application either intravenously, subcutaneously or intramuscularly. The compound is a potent immunopotentiating agent with a daily dosage in the range of about 1 to 100 .mu.g/kg of body weight per day for intravenous administration. Obviously, the required dosage will vary with the particular condition being treated, the severity of the condition and the duration of the treatment.
In U.S. Pat. No. 4,148,788, the present inventor described the synthesis of thymosin .alpha..sub.1 by solid-phase peptide synthesis and by fragmentation condensation. These processes both used tert. butyloxycarbonyl (Boc) as the protecting group for the .alpha.-amino acid during the coupling reactions of the C-terminal amino acid to the solid support resin and for the coupling of each successive amino acid.
The Boc group has been the preferred amino protecting group for solid-phase peptide synthesis in view of its stability to the condensation (coupling) conditions, its ease of removability without destruction of the peptide bonds or racemization of chiral centers in the peptide chain, and its low cost. This preference for the Boc protective group is clearly manifested in the patent and general literature by the almost exclusive use of Boc as the protective group for protecting alpha-amino acids.
For instance, G. Barany and R. B. Merrifield in The Peptides, Analysis, Synthesis, Biology, Vol. 2, "Special Methods in Peptide Synthesis", Part A. Ed. By E. Gross and J. Meienhofer, Academic Press, Inc., New York, 1980, state in Chapter 1, Solid-Phase Peptide Synthesis at pages 101-102, that "[t]he tert-butyloxycarbonyl (Boc) group . . . is by far the most widely used functionality for .alpha.-amino protection in solid-phase peptide synthesis." Other tert-alkyl urethane forming .alpha.-amino protecting groups mentioned by the authors include tert-amyloxycarbonyl (Aoc), adamantyloxycarbonyl (Adoc), and 1-methylcyclobutyloxycarbonyl (Mcb). This text also describes 10 different combinations of inorganic (protic or Lewis) and organic (including carboxylic) acids, in solvent (usually anhydrous) mixtures. Among these combinations are neat anhydrous trifluoroacetic acid (TFA) and TFA-CH.sub.2 Cl.sub.2 in ratios (v/v) from 1:4 to 1:1, 25.degree. C., 20-30 minutes.
Under the heading "3. Other Amino-Protecting Groups Removable by Acidolysis" pages 106-107, Barany and Merrifield report that the benzyloxycarbonyl (Z) group, which is removed by strong acids such as HBr in acetic acid, was the first group examined for solid-phase synthesis, but that
On the other hand, the authors also report on work by others using furfuryloxycarbonyl (Foc) and p-methoxybenzyloxycarbonyl (Moz) groups. They state at page 107 that these
In the aforementioned U.S. Pat. No. 4,148,788 only Boc is disclosed as the .alpha.-amino protecting group although as the protecting group for the .omega.-amino group of lysine (Lys) the benzyloxycarbonyl (Z) protecting group is used and other .omega.-amino protecting groups are disclosed, including benzyloxycarbonyl substituted in the aromatic ring, such as by 4-chloro, 2-bromo, 4-bromo, 2,4-dichloro, 4-nitro, 4-methoxy, 3,5-dimethoxy, 4-methyl, 2,4,6-trimethyl, 4-phenylazo, 4-(4-methoxyphenylazo), 2-(N,N-dimethylcarbonamido), 4-dihydroxyboryl, and 2-nitro-4,5-dimethoxy, urethane type protecting groups, such as 4-toluenesulfonylethyloxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and related base cleavable groups, 5-benzisoxazolylmethyleneoxycarbonyl, methylthio- and methylsulfonylethyloxycarbonyl, isonicotinyloxycarbonyl, haloethyloxycarbonyl, diisopropylmethyloxycarbonyl, benzhydryloxycarbonyl, isobornyloxycarbonyl, dinitrodiphenylmethyloxycarbonyl, tert. butyloxycarbonyl, tert. amyloxycarbonyl, adamantyloxycarbonyl, cyclopentyloxycarbonyl, and others; acyl groups, such as formyl, trifluoroacetyl, phthaloyl, benzenesulfonyl, and others; and aryl-lower alkyl groups, such as diphenylmethyl and triphenylmethyl.
In the recently published European Patent Application 0 200 404 published Nov. 5, 1986, and corresponding to U.S. application Ser. No. 722,218, filed Nov. 4, 1985, now abandoned, and to continuation-in-part application Ser. No. 849,835, filed Apr. 9, 1986, now U.S. Pat. No. 4,855,407, issued Aug. 8, 1989, the present applicant disclosed an improved solid-phase peptide synthesis of thymosin .alpha..sub.1, using methylbenzhydrylamine (MBHA) resin as the solid support and hydrogen bromide (HBr) with trifluoroacetic acid (TFA) and anisole and thioanisole as the cleavage and deprotection agents. Here too, Boc is disclosed as the preferred, and is used as the, alpha-amino protecting group. Other .alpha.-amino protecting groups are mentioned, including benzyloxycarbonyl (Z), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, 1,1-dimethyl-3,5-dimethyloxybenzyloxycarbonyl (Ddz), o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl and 9-fluorenylmethyloxycarbonyl.
The patent literature, disclosing solid-phase peptide synthesis of peptides, other than thymosin .alpha..sub.1, also substantially exclusively exemplifies Boc as the alpha,amino protecting group, notwithstanding general disclosures of other protecting groups, such as benzyloxycarbonyl (Z) and substituted Z groups, including, for example, those mentioned above as protecting group for .omega.-amino functional groups.
On the other hand, for classical liquid phase (solution) peptide synthesis reactions, the benzyloxycarbonyl (Z) and Boc alpha-amino protecting groups are used with about equal frequency. Thus, while acidolytic cleavage of alpha-amino protecting groups has been the subject of considerable research, the general conclusion in the art has been that the acid sensitivities of the Boc or 4-methoxy substituted Z, i.e. 4-methoxybenzyloxycarbonyl (Moz), protecting groups are roughly equal. So far as the applicant is aware, however, no precise or rigorous comparison has ever actually been conducted to test the relative acid sensitivities of Boc and Moz in solid-phase peptide synthesis.
In the aforementioned U.S. application Ser. Nos. 722,218 and 849,835, U.S. Pat. No. 4,855,407 and the corresponding published application 0,200,404 the advantages of using hydrogen bromide as the acid cleavage and deprotection agent in place of the more highly corrosive hydrogen fluoride were described.
However, the use of HBr does present the drawback that, being a gas, it is brought into contact with the resin-bound protected thymosin .alpha..sub.1 by bubbling the gaseous HBr into a solution of the resin-bound protected peptide in, usually, trifluoroacetic acid. The bubbling procedure is not only slow but also requires pressurized gas tanks and still requires some degree of skill in its use. Therefore, it would be desirable to find a liquid acid cleavage agent which does not present the corrosion problems inherent to HF and can be used with ordinary laboratory glassware or plastic yet is at least as highly efficient and selective in the acid cleavage and deprotection of the resin-bound protected thymosin .alpha..sub.1 peptide.
Accordingly, it is an object of this invention to provide an improved solid-phase synthesis of thymosin .alpha..sub.1 in high yield and high purity.
Another object of the invention is to provide an improved solid-phase synthesis of thymosin .alpha..sub.1 which can be easily and efficiently carried out without requiring highly skilled technicians and which avoids the use of gaseous reactants for the cleavage and deprotection of the resin-bound protected thymosin .alpha..sub.1 peptide.
It is another object to provide a solid-phase synthesis for thymosin .alpha..sub.1 which is capable of large scale synthesis using standard equipment and apparatus.
These and other objects of the invention which will become more apparent upon review of the following detailed description and preferred embodiments, including the accompanying drawing figures, are provided by the solid-phase peptide synthesis for production of thymosin .alpha..sub.1 in which during synthesis the nitrogen of the alpha-amino group (N.sup..alpha.) of each amino acid is bonded to 4-methoxybenzyloxycarbonyl (Moz) and cleavage is carried out using greatly reduced quantity of trifluoroacetic acid (TFA).
According to another aspect of the invention, in a preferred embodiment thereof, the step of cleavage and deprotection of the resin-bound protected thymosin .alpha..sub.1 peptide is carried out using trifluoromethane sulfonic acid (TFMSA), by adding the TFMSA, preferably together with anisole and thioanisole, to a suspension of the resin-bound protected peptide in, preferably, trifluoroacetic acid.
In accordance with this invention, thymosin .alpha..sub.1 or a biologically active analog or fragment thereof is prepared by solid-phase peptide synthesis by the steps of
Preferably, the resin support in step (b) is methylbenzhydrylamine resin and step (g) is carried out with hydrogen bromide or trifluoromethane sulfonic acid.